The central goal of this work is the development of an innovative, real-time, nanoscale, biochemical mapping methodology enabled through the fabrication of a multiscale fluidic interface in registration with patterned ensembles of cultured retinal pigment epithelial (RPE) cells that will permit exquisite spatiotemporal resolution of gene expression in the apical and basolateral milieus, with high molecular detection sensitivity (on the order of single secreted molecules), and broad dynamic range. The unique enabling concept for this work that distinguishes it from the current state-of-the- art is the marriage of two emerging technologies:1) a multiscale nanoplatform (NP), fabricated using a porous membrane for cell attachment and culture, together with soft lithography to create addressable nanofluidic arrays within the microscale chemostat (i.e., a bioreactor with a defined chemical environment), and 2) the use of multiphase fluidics involving water-in-oil droplet formation as programmable, discrete, nanoscale reaction volumes capable of highly localized dosing and sampling from RPE cells in situ. The RPE plays a critical role in the maintenance of the subretinal microenvironment and is essential to normal retinal health and photoreceptor function. Age-related macular degeneration (AMD) is associated with degeneration of the RPE layer, and abnormal regulation of pro- and antiangiogenic molecules such as vascular endothelial growth factor (VEGF), IGF-1, and matrix metalloproteinases. Understanding when and how regulation of these molecules becomes pathologic will require an improved understanding of their normal dynamic flux and how they are induced by pathological angiogenesis. Our platform and model combine a novel, translational, molecular approach to define spatiotemporal gene expression with the ability to manipulate the apical and basal microniches to understand the dynamic regulation of angiogenesis by the RPE. Our Specific Aims are, 1) to compare cells derived from normal, aging, and diseased RPE tissue layers to establish the steady-state behavior of polarized RPE cells on-chip, including constitutive expression and secretion of pro- and antiangiogenic molecules, and the expression of key antioxidant and VEGF-related genes, and 2) to capture spatiotemporal profiles of secreted proangiogenic proteins under hypoxic and oxidative stress (OS). Our technology, together with the results of our studies, will provide a new understanding of the mechanisms driving proangiogenic gene expression in chronic versus pulsatile oxidative and hypoxic stress in the retina that has not previously been possible. It will enable the development of real-time spatiotemporal maps of the molecular signaling in OS and hypoxia and a deeper understanding of the regulation of genes associated with pathological neovascularization in AMD.